RBL-2H3 cell, a cultured rat mast cell line, generates the cytokines, TNFalpha and IL-6, at physiologically active concentrations, in response to stimulation with antigen. Both production and release of these cytokines are dependent on cell stimulation and both appear to be distinct regulated processes. Studies with various stimulants and inhibitors have indicated that production of TNFalpha is dependent on mobilization of Ca2+, activation of protein kinase C and a third synergising signal; secretion of the newly formed TNFalpha, which is dependent on Golgi processing, is regulated by mobilization of Ca2+ and activation of protein kinase C. Although mobilization of Ca2+ or the activation of protein kinase C alone will stimulate some production of TNFalpha both signals are necessary for secretion of newly formed cytokine. Of the various stimulants studied, antigen and the endotoxin, lipopolysaccharide, were uniquely potent stimulants of TNF production when stimulants were tested at concentrations that were optimal for release of preformed secretory granules. Other stimulants included the protein kinase C activator, phorbol 12-myristate 13-acetate, Ca2+- ionophore and, in cells transfected with the gene for the muscarinic m1 receptor, carbachol. In contrast to the above, the anti-inflammatory cytokine, TGFbeta, was found to be constitutively produced and secreted via Golgi and these processes were not further influenced by mobilization of Ca2+ or activation of protein kinase C as indicated from studies with the aforementioned stimulants. Therefore, all three cytokines are processed via Golgi but their release may be either constitutive or subject to receptor-mediated signals.